LC8 enhances 53BP1 foci through heterogeneous bridging of 53BP1 oligomers
- Oregon State University, United States
- The University of Texas Health Science Center at San Antonio, United States
Abstract
53BP1 is a key player in DNA repair and together with BRCA1 regulate selection of DNA double strand break repair mechanisms. Localization of DNA repair factors to sites of DNA damage by 53BP1 is controlled by its oligomerization domain (OD) and binding to LC8, a hub protein that functions to dimerize >100 clients. Here we show that 53BP1 OD is a trimer, an unusual finding for LC8 clients which are all dimers or tetramers. As a trimer, 53BP1 forms a heterogeneous mixture of complexes when bound to dimeric LC8 with the largest mass corresponding to a dimer-of-trimers bridged by 3 LC8 dimers. Analytical ultracentrifugation and isothermal titration calorimetry demonstrate that only the second of the three LC8 recognition motifs is necessary for a stable bridged complex. The stability of the bridged complex is tuned by multivalency, binding specificity of the second LC8 site, and the length of the linker separating the LC8 binding domain and OD. 53BP1 mutants deficient in bridged species fail to impact 53BP1 focus formation in human cell culture studies, suggesting that the primary role of LC8 is to bridge 53BP1 trimers which in turn promotes recruitment of 53BP1 at sites of DNA damage. We propose that the formation of higher-order oligomers of 53BP1 explains how LC8 elicits an improvement in 53BP1 foci and affects the structure and functions of 53BP1.
Data availability
All data generated or analysed during this study are included in the manuscript; source data files have been provided for Figures 1-4, 6-7
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Funding
National Institute of General Medical Sciences (R01GM141733)
- Elisar J Barbar
National Institute of General Medical Sciences (R35GM137798)
- Justin WC Leung
National Cancer Institute (R01CA244261)
- Justin WC Leung
American Cancer Society (RSG-20-131-01-DMC)
- Justin WC Leung
ARCS Oregon Chapter (Graduate Student Fellowship)
- Jesse Howe
National Institute of Health (1S10OD018518)
- Elisar J Barbar
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2025, Howe et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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LC8 enhances 53BP1 foci through heterogeneous bridging of 53BP1 oligomers
eLife 14:e102179.
https://doi.org/10.7554/eLife.102179
